The heart is dependent on intercellular communication through gap junction channels for action potential propagation and normal electrical conduction and contraction. We have demonstrated that the heart contains multiple gap junction proteins (connexins, Cx), including the extensively studied Cx43 (which is abundant in ventricular myocardium) and the more recently identified Cx40 and Cx45 (which exhibit unique patterns of expression within cardiac tissues and form gap junction channels with distinct conductance and regulatory properties). The overall goal of our studies is to understand the regulation of cardiac intercellular communication and, specifically, the roles of the multiple channel proteins, They likely contribute to this regulation through differential protein regulation (including phosphorylation and turnover), differential connexin gene expression, nd connexin-specific channel properties. Two Specific Aims are proposed; 1. We will conduct biochemical and molecular studies to determine and compare regulation of the cardiac connexin proteins and mRNAs. We will define the phosphorylation sites in Cx45 and their contribution to channel gating, connexin stability, and gap junction assembly. We will determine the turnover dynamics of the cardiac connexins and the mechanism of degradation of Vx43 and Cx45 (in particular the role of ubiquitin-dependent proteolysis). We will determine the structure and sequence of the Cx40 gene and elements important in the expression of Cx40 mRNA. 2. We will determine and compare the conductance and permeability properties of the cardiovascular connexins as expressed in a stably transfected, communication-deficient cell line (N2A) and analyzed by double whole cell patch clamp methods. We will determine the monovalent cation and anion selectivities, differential molecular permeabilities to fluorescent dyes and second messengers, and abilities to form heterotypic channels for all cardiac connexins. Then, we will use the expression of selected chimeric or mutant connexin constructs to determine residues important in channel selectivity. Our studies and those of others have shown that the pattern of expression of gap junctions is related to the different anisotropic and conductive properties of different cardiac tissues and that derangements of gap junctions are present in various pathological regions, including ones that may predispose to the development of reentrant arrhythmias. Therefore, a molecular description of the regulation of gap junction mediated intercellular communication may have profound implications for understanding normal conduction and perhaps controlling arrhythmias.